Characterization of Extracellular Vesicles Produced byAspergillus fumigatus Protoplasts

Juliana Rizzo,a,b Thibault Chaze,c Kildare Miranda,d,e Robert W. Roberson,f Olivier Gorgette,g Leonardo Nimrichter,a

Mariette Matondo,c Jean-Paul Latgé,b,h Anne Beauvais,b Marcio L. Rodriguesa,i

aInstituto de Microbiologia Paulo de Góes (IMPG), Universidade Federal do Rio de Janeiro, Rio de Janeiro, BrazilbDepartment of Mycology, Institut Pasteur, Paris, FrancecPlateforme Protéomique, Unité de Spectrométrie de Masse pour la Biologie (MSBio), Centre de Ressources et Recherches Technologiques (C2RT), USR 2000 CNRS,Institut Pasteur, Paris, France

dInstituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, BrazileCentro Nacional de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, BrazilfSchool of Life Sciences, Arizona State University, Tempe, Arizona, USAgPlateforme de Microscopie Ultrastructurale, Imagepole, Institut Pasteur, Paris, FrancehSchool of Medicine, University of Crete, Heraklion, Crete, GreeceiInstituto Carlos Chagas, Fundação Oswaldo Cruz, Curitiba, Brazil

Anne Beauvais and Marcio L. Rodrigues share co-senior authorship of this article.

ABSTRACT Extracellular vesicles (EVs) are membranous compartments produced byyeast and mycelial forms of several fungal species. One of the difficulties in perceiv-ing the role of EVs during the fungal life, and particularly in cell wall biogenesis, iscaused by the presence of a thick cell wall. One alternative to have better access tothese vesicles is to use protoplasts. This approach has been investigated here withAspergillus fumigatus, one of the most common opportunistic fungal pathogensworldwide. Analysis of regenerating protoplasts by scanning electron microscopyand fluorescence microscopy indicated the occurrence of outer membrane projec-tions in association with surface components and the release of particles with prop-erties resembling those of fungal EVs. EVs in culture supernatants were characterizedby transmission electron microscopy and nanoparticle tracking analysis. Proteomicand glycome analysis of EVs revealed the presence of a complex array of enzymesrelated to lipid/sugar metabolism, pathogenic processes, and cell wall biosynthesis.Our data indicate that (i) EV production is a common feature of different morpho-logical stages of this major fungal pathogen and (ii) protoplastic EVs are promisingtools for undertaking studies of vesicle functions in fungal cells.

IMPORTANCE Fungal cells use extracellular vesicles (EVs) to export biologically ac-tive molecules to the extracellular space. In this study, we used protoplasts of Asper-gillus fumigatus, a major fungal pathogen, as a model to evaluate the role of EV pro-duction in cell wall biogenesis. Our results demonstrated that wall-less A. fumigatusexports plasma membrane-derived EVs containing a complex combination of pro-teins and glycans. Our report is the first to characterize fungal EVs in the absence ofa cell wall. Our results suggest that protoplasts represent a promising model forfunctional studies of fungal vesicles.

KEYWORDS Aspergillus fumigatus, conidia, protoplasts, extracellular vesicles,Aspergillus

In the Aspergillus genus, 90% of all infections resulting in human aspergillosis arecaused by Aspergillus fumigatus, which is the most prevalent mold pathogen in

immunocompromised patients (1). A. fumigatus has a multifactorial pathogenic arsenal,

Citation Rizzo J, Chaze T, Miranda K, RobersonRW, Gorgette O, Nimrichter L, Matondo M,Latgé J-P, Beauvais A, Rodrigues ML. 2020.Characterization of extracellular vesiclesproduced by Aspergillus fumigatus protoplasts.mSphere 5:e00476-20. https://doi.org/10.1128/mSphere.00476-20.

Editor Aaron P. Mitchell, University of Georgia

Copyright © 2020 Rizzo et al. This is an open-access article distributed under the terms ofthe Creative Commons Attribution 4.0International license.

Address correspondence to Anne Beauvais,anne.beauvais@pasteur.fr, or Marcio L.Rodrigues, marcio.rodrigues@fiocruz.br.

Received 20 May 2020Accepted 22 July 2020Published

RESEARCH ARTICLEMolecular Biology and Physiology

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which allows this organism to successfully establish disease in different hosts (1–4).Aspergillosis begins with inhalation of asexual conidia followed by fungal morpholog-ical transition in the absence of a proper immunological response (1).

Fungi, as seen with many other eukaryotic and prokaryotic organisms, produceextracellular vesicles (EVs) (5–8). EVs were first described in the yeast-like pathogenCryptococcus neoformans (9). Subsequent studies demonstrated EV production in yeastforms of C. gattii, Histoplasma capsulatum, Candida albicans, C. parapsilosis, Sporothrixschenckii, S. brasiliensis, Paracoccidioides brasiliensis, P. lutzii, Malassezia sympodialis,Saccharomyces cerevisiae, Pichia fermentans, and Exophiala dermatitidis (10–17). Infilamentous fungi, the presence of EVs was described previously in the phytopathogensAlternaria infectoria (18) and Fusarium oxysporum f. sp. vasinfectum (19), in the der-matophyte Trichophyton interdigitale (20), and in the emerging human pathogenRhizopus delemar (21). Recently, it was also reported that mycelial forms of A. fumigatusproduce EVs (22).

A major difficulty in directly addressing the physiological roles of EVs is the lack ofunderstanding of their intracellular biogenesis and of the mechanisms underlying cellwall crossing. An original approach for studying the generation of EVs would be to useprotoplasts and look at their active release in the absence of a cell wall. In fact,protoplasts might represent a promising model for the study of EVs, as alreadysuggested by Gibson and Peberdy 50 years ago, who chose the name “subprotoplasts”for the vesicle-like particles budding from the plasma membrane of A. nidulans cells(23).

Our primary goal in the present work was to search for EVs produced by protoplastsof A. fumigatus germinating conidia. Our results revealed the presence of typical EVs inprotoplast supernatants incubated under different conditions. EV cargo was directlyinfluenced by the experimental conditions under which A. fumigatus was incubated.Our report provides experimental evidence that fungal EVs are produced not only bythe mycelial morphological stage of A. fumigatus, but also by protoplasts of germinat-ing conidia, especially during cell wall regeneration. To our knowledge, this is the firstdemonstration that protoplasts may represent a useful model to analyze the produc-tion and role of EVs in the absence of any cell wall in an experimental setting similarto the ones which allowed the study of exosomes produced by mammalian cells (24).

RESULTSObservation of outer particles resembling EVs in protoplasts of A. fumigatus

germinating conidia. To experimentally overcome the difficulties of detecting EVs insitu due to the presence of a thick cell wall, we adopted an experimental model usingprotoplasts. Fungal cells lacking cell walls were obtained by enzymatic digestion witha lysing enzyme from Trichoderma harzianum (Glucanex), which hydrolyzes cell wallcomponents for protoplast preparation (25). These cells can reconstruct their walls inosmotically stabilized media containing 0.6 M KCl, glucose, nitrogen, and salts (26–30).Incubation of protoplasts in 0.6 M KCl only, on the other hand, impairs cell wallsynthesis (A. Beauvais and T. Fontaine, unpublished data). In this context, we firstcompared the morphological aspects of freshly obtained A. fumigatus protoplasts withcell wall-regenerating cells by scanning electron microscopy (SEM). Under both exper-imental conditions, we observed �50-nm-diameter extracellular structures in an ap-parent association with the fungal surface (Fig. 1). When the protoplasts were incu-bated under conditions of cell wall synthesis, the outer particles were more numerous,and a fibril-like network was observed.

To analyze these vesicle-like particles in intact protoplasts, we checked the mem-brane organization of freshly prepared protoplasts, protoplasts undergoing cell wallsynthesis for 2 h (medium with the osmotic stabilizer KCl and nutrients), and proto-plasts incubated for 2 h under the starvation condition (in KCl only). Incubationoutcomes were monitored by staining the protoplasts with an anti-glucan antibodyand observation by fluorescence microscopy. Membranes were stained with the lipo-philic dye DiI. A. fumigatus protoplasts manifested the complex membrane distribution

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that is typically observed in most eukaryotic cells (Fig. 2A). As expected, glucan wasdetected at the background levels in freshly prepared protoplasts and in protoplastsincubated in KCl alone. Under conditions of cell wall synthesis, surface glucan wasunequivocally detected. A detailed analysis of the relationship between membranestaining and glucan detection in these cells revealed several regions of membraneprojection to the outer space (Fig. 2B). Under nonregenerating conditions, no glucanwas detected (Fig. 2C). During induction of cell wall synthesis, the projected regionswere closely associated with glucan detection, and, in fact, the polysaccharide wasapparently surrounded by the membranous compartments (Fig. 2D). The occurrence ofmembrane projections in protoplasts incubated under nonregenerating or cell wallsynthesis conditions was confirmed by superresolution SEM (Fig. 2E and 2F, respec-tively).

Protoplasts of A. fumigatus germinating conidia release EVs. Fungal prepara-tions used for both qualitative and quantitative EV analyses were adjusted to 108

protoplasts/ml, and cell viability was in the 87% to 93% range throughout all of theexperiments. Supernatants obtained from protoplasts incubated under regeneratingand nonregenerating conditions were fractionated by ultracentrifugation, and theresulting pellets were analyzed by transmission electron microscopy (TEM). Membra-nous structures with a typical bilayer and with the morphological aspects and dimen-sions previously observed for fungal EVs were isolated from protoplast supernatants(Fig. 3A, B, E, and F). Regenerating protoplasts produced EVs that were apparentlyassociated with fibrillar material (Fig. 3G and H; see also Fig. S1 in the supplementalmaterial). Most of the vesicles were in the 200-nm-diameter range, and this visualperception was confirmed by nanoparticle tracking analysis (NTA), which revealed amajor peak of vesicle detection in the 150-nm-diameter range (Fig. 3I and J). EVsobtained from control or regenerating protoplasts had very similar diameter distribu-tion profiles.

EVs are more abundantly detected in the supernatants of regenerating pro-toplasts. We quantified EV production in the experimental systems explored in ourstudy by different approaches. First, independent replicates were submitted to quan-titative NTA. This analysis revealed that the EV/cell ratios were at the background levelsfor the samples from freshly prepared protoplasts (Fig. 4A). Even though some EVs werereleased over time in the supernatants of control protoplasts, their number was

FIG 1 Morphological aspects of freshly prepared and cell wall-regenerating protoplasts. Fresh protoplasts (A to C)and cell wall-regenerating cells (D to F) are shown under conditions of increased magnification by SEM. Panels Cand F represent magnified views of the boxed areas in panels B and E, respectively. The magnified views suggestedthe occurrence of outer particles with properties compatible with EVs (white arrows). Under cell wall-regeneratingconditions, a fibril-like network was more abundantly detected (yellow arrow). Scale bars represent 5 �m in panelsA and D, 2 �m in panels B and E, and 1 �m in panels C and F. At least 50 cells were analyzed, and the results arerepresentative of at least two independent experiments producing similar morphological profiles. Similar analysesusing superresolution SEM produced similar results (data not shown).

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drastically increased in the supernatant of regenerating protoplasts. The NTA dataagreed with the quantification of the sterols in the EV-containing supernatants (Fig. 4B).This increase in the sterol levels was not associated with an enhancement in the totalamount of the protoplast sterols occurring during cell wall regeneration (data notshown).

Glycan components of A. fumigatus EVs. The molecular composition of the EVsreleased by the protoplasts was investigated. Since galactosaminogalactan (GAG) is amarker of polysaccharide secretion by the A. fumigatus mycelium (4), we first showedthe presence of this polysaccharide during protoplast regeneration (Fig. 5A). We thenproved that GAG was present in EVs isolated from protoplasts incubated under theconditions of cell wall regeneration and was absent in control vesicles (Fig. 5B). Theseresults agreed with the compositional analysis of the carbohydrate units of EVs.Glucosyl (Glc), mannosyl (Man), and galactosyl (Gal) units were detected in EV prepa-rations obtained from both cell wall-regenerating and nonregenerating protoplasts(Fig. 5C). N-Acetyl-galactosaminyl (GalNAc) residues, which are markers of GAG, were

FIG 2 Membrane projections in A. fumigatus protoplasts. (A) Freshly purified protoplasts were stained with DiI, a lipophilicdye (red fluorescence). Cell wall staining with an anti-glucan antibody was at the background levels. Similar results wereobserved for protoplasts incubated under nonregenerating conditions. During cell wall regeneration (2 h), glucan staining(green fluorescence) was abundant at the cell surface (asterisk). (B) Detailed analysis of nonregenerating and cellwall-regenerating cells revealed an association between glucan staining and outer membrane projections only in cellwall-regenerating protoplasts (90 min of incubation). (C and D) Enhanced views of the boxed areas (numbered 1 to 4) offungal cells in the absence of cell wall synthesis and under cell wall-regenerating conditions, respectively. (E and F) Adetailed view of the surface of protoplasts provided by superresolution SEM confirmed the occurrence of outer particles(white arrows) budding from the plasma membrane in nonregenerating protoplasts (E) and regenerating (2 h) protoplasts(F). Fibrillar material closely associated with the outer membrane projection was uniquely detected during cell wallregeneration (F, yellow arrow). At least 10 cells were analyzed, and the results are representative of two independentexperiments producing similar morphological profiles.

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found only in regenerating protoplasts. In addition, Glc levels were significantly in-creased in EV samples from these cells compared to nonregenerating protoplasts(P � 0.004). N-Acetylglucosamine (GlcNAc) residues were absent in all samples.

Proteomic analysis of EVs. Proteomic analysis revealed only 142 proteins in EVsproduced by fresh protoplasts, contrasting with the detection of 2,056 proteins in

FIG 3 Analysis of EVs obtained from A. fumigatus protoplasts. (A to H) Protoplast EVs were analyzed by regularTEM (A, B, E, and F) or after negative staining (C, D, G, and H). Independent illustrations of each condition areshown for each technique. Under conditions stimulating cell wall synthesis, fibril-like structures associated withEVs were observed (G and H, arrows). The results are representative of at least two independent experimentsproducing similar morphological profiles. Scale bars correspond to 100 nm. (I and J) NTA of isolated vesiclesdemonstrated similar distributions of EVs in the 50-to-300-nm-diameter range, independently of the conditionof incubation of the protoplasts. NTA was repeated twice, producing similar results.

FIG 4 EV quantification during the cell wall synthesis process in A. fumigatus protoplasts. (A) Quanti-tative NTA of EVs produced by freshly prepared protoplasts and from protoplasts incubated underconditions of cell wall regeneration or nonregeneration. (B) Determination of sterol concentration in EVsobtained from supernatants of fresh protoplasts, nonregenerating protoplasts, and protoplasts incu-bated under cell wall-regenerating conditions. In panels A and B, values are reported as means �standard deviations of results obtained from at least two and five independent experiments, respectively.Paired comparisons were statistically analyzed using the t test tool in GraphPad Prism 6 software.

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FIG 5 Analysis of glycan synthesis during cell wall regeneration in A. fumigatus conidial protoplasts. (A) Membrane and GAG staining in A. fumigatus protoplasts.All cells were efficiently stained with DiI (red fluorescence). During cell wall synthesis (2 h), GAG was detected in association with the fungal surface. The scalebar corresponds to 5 �m. (B) Serological detection of GAG (ELISA) in EVs obtained from protoplasts. Positive reactions with a GAG-binding antibody wereobserved only in EVs obtained during cell wall synthesis. (C) Gas chromatography-mass spectrometry (GC-MS) analysis of sugar units of EVs. In agreement withan involvement of EVs in cell wall synthesis, GalNAC (a GAG component) was observed only in EVs obtained from protoplasts during cell wall regeneration.The increased detection of Glc during cell wall synthesis (2 h of germination) is consistent with the presence of EV-associated glucans. The results arerepresentative of two independent replicates producing similar profiles.

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vesicles from regenerating protoplasts (see Table S1 in the supplemental material). All142 of the EV proteins detected in fresh protoplasts were found in samples obtainedunder cell wall regeneration conditions. Although EVs were more abundantly detectedin the regenerating protoplasts, the qualitative protein composition of the nonregen-erating protoplast was similar (Table S1).

The predicted GO classification of all EV-related proteins identified numerous terms(Fig. 6). As previously described for several fungal EVs (11, 14, 22, 31), the shared GOterms corresponded to proteins involved in a wide range of processes of fungalphysiology. Most of the biological processes (680 GO terms) were common to both theregenerating and nonregenerating conditions. A minor fraction of biological processes(50 GO terms) were specifically found in the cell wall-regenerating system. We used theUniProt (https://www.uniprot.org/) and AspGd (http://www.aspgd.org/) databases tospecifically analyze proteins related to cell wall assembly under each of the sets ofexperimental conditions used in this study (Table 1). We detected several proteinsrelated to (i) cell wall synthases, including the �1,3 glucan synthase Fks1 and its GTPaseactivator Rho1 (32), �1,3 glucan synthases Ags (33), chitin synthases (34), Ktr manno-syltransferases involved in the synthesis of galactomannan (35), and enzymes belong-ing to the GAG biosynthetic pathway (Ugm1, Gt4c) (36); (ii) cell wall remodelingenzymes, including Gel and Bgt glucosyltransferases involved in the elongation andbranching of the �1,3 glucan (37); and (iii) some enzymes involved in mannosylation,including multienzyme complexes mannan polymerase I and II involved in the synthe-sis of one of the two conidial mannans (Mnn proteins, Van1, Och1) (38) and PmtO-mannosyltransferases involved in the 0-mannosylation of cell wall remodeling en-zymes (39). Other cell wall-related proteins, including Mp1, PhiA, and MidA; glycosyl-phosphatidylinositol (GPI)-anchored proteins, including Ecm33; mannosyltransferases(Alg2, Och1and MnnII); and the putative glycan biosynthesis protein Pig1, were also

FIG 6 Proteomic analysis of EVs obtained from supernatants of A. fumigatus protoplasts. TreeMap views of all biological processes with which vesicular proteinswere associated are presented. Panel A shows the biological processes common to regenerating and nonregenerating conditions. Panel B shows the processesthat were exclusively found under conditions of cell wall regeneration. Rectangular areas reflect the P value of enrichment of GO terms in the Aspergillusdatabase. GO terms are gathered under summarized terms using the REVIGO tool (77). ncRNA, noncoding RNA. Major cellular processes are specified in panelsA and B. Subclasses of each cellular process are listed on the figure’s right side.

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TABLE 1 Cell wall-associated proteins found in A. fumigatus EVs produced by protoplastsa

Condition UniProt annotationUniProtno. Accession no.

Standardname(s)

Cell wall synthesis only Probable glucan endo-1,3-beta-glucosidase EglC B0XXF8 AFUB_048180 Bgt2Probable beta-glucosidase E BglE B0YD91 AFUB_094720 Exg21Filament-forming protein (Tpr/p270), putative B0XM26 AFUB_001640Cell wall biogenesis protein Mhp1, putative B0XR76 AFUB_012380O-Methyltransferase B0XVZ1 AFUB_0335001,4-Alpha-glucan branching enzyme B0Y0Q4 AFUB_058160Alpha-1,2-mannosyltransferase (Alg2), putative B0Y1U9 AFUB_060920 Alg2Alpha-1,6-mannosyltransferase subunit (Och1), putative B0Y410 AFUB_056120 Och1Glycan biosynthesis protein (PigL), putative B0YAG1 AFUB_084550 PigLAlpha-1,2-Mannosidase B0Y765 AFUB_072720Alpha-1,2-mannosyltransferase, putative B0Y1T7 AFUB_060800 MnnIIAlpha-N-acetylglucosamine transferase B0YA98 AFUB_083900

Cell wall repression only Mannosylphosphorylation protein (Mnn4), putative B0XN98 AFUB_004200 Mnn4GPI-anchored cell surface glycoprotein, putative B0Y1D8 AFUB_059930Chitin synthase activator (Chs3), putative B0Y9Q8 AFUB_081930 Chs3Mannosyltransferase PMTI B0YA13 AFUB_083000 Pmt4Probable glucan endo-1,3-beta-glucosidase BtgC B0Y429 AFUB_056310 Bgt3Cell wall proline-rich protein, putative B0XRJ9 AFUB_012940Glycosyl hydrolase, putative B0XYB1 AFUB_040280

Both cell wall synthesis andcell wall repression

Chitin synthase, putative B0XTD9 AFUB_029070 CsmB

Chitin synthase ChsE B0XTE0 AFUB_029080 ChsEChitin synthase B0XTK9 AFUB_018960 ChsAChitin synthase activator (Chs3), putative B0XZ75 AFUB_043410 Chs3Chitin synthase B0XZY5 AFUB_034810 ChsGChitin synthase F B0Y9Q7 AFUB_081920 ChsFClass V chitinase, putative B0YBH2 AFUB_092800Chitin biosynthesis protein (Chs5), putative B0YDJ8 AFUB_095840 Chs5Chitin biosynthesis protein (Chs7), putative B0XQX5 AFUB_011500 Chs7Alpha-1,3-glucan synthase, putative B0XNF7 AFUB_014990 Ags1/Ags2/Ags3Alpha-1,3-glucan synthase, putative B0XX26 AFUB_047490 Ags1/Ags2/Ags31,3-Beta-glucan synthase catalytic subunit FksP B0Y8S7 AFUB_078400 Fksp/Fks1Cell wall protein PhiA B0Y004 AFUB_045170 Aspf34Cell wall biogenesis protein phosphatase Ssd1, putative B0XQR1 AFUB_010850 Ssd1Cell wall protein, putative B0XXP9 AFUB_038170 MidAGPI-anchored cell wall protein, putative B0Y688 AFUB_066060GPI-anchored cell wall organization protein Ecm33 B0Y5M3 AFUB_063890 Ecm33GPI-anchored protein, putative B0YDG5 AFUB_095500Cell wall integrity signaling protein Lsp1, putative B0Y7E0 AFUB_073480 Pil1Cell wall serine-threonine-rich galactomannoprotein Mp1 B0YEP2 AFUB_099880 Mp11,3-Beta-glucanosyltransferase Gel1 B0XT72 AFUB_018250 Gel11,3-Beta-glucanosyltransferase Gel4 B0XVI5 AFUB_022370 Gel4Mannan endo-1,6-alpha-mannosidase B0XXF1 AFUB_048110Alpha-1,6-mannosyltransferase subunit (Mnn9), putative B0XTG8 AFUB_018530 Mnn9Dolichol-phosphate mannosyltransferase, putative B0XXW0 AFUB_038750Protein mannosyltransferase 1 B0XYZ3 AFUB_042600 Pmt1Alpha-1,2-mannosyltransferase (Kre2), putative B0Y0S4 AFUB_058360 Ktr1Alpha-1,2-mannosyltransferase (Kre5), putative B0Y1C4 AFUB_059750 Ktr7Alpha-1,2-mannosyltransferase (Ktr4), putative B0Y2F5 AFUB_051270 Ktr4Alpha-1,6-mannosyltransferase subunit, putative B0Y6R0 AFUB_067830 Mnn11UDP-glucose 4-epimerase B0Y0S6 AFUB_058380 Uge5UDP-glucose:glycoprotein glucosyltransferase, putative B0XTX7 AFUB_019450Glycosyl transferase, putative B0XYK7 AFUB_041250 Gt4b/Gt4cGlycosyl transferase, putative B0YAG3 AFUB_084570 Och3Glycosyl transferase, putative B0XZM8 AFUB_044600N-Glycosyl-transferase B0Y5M8 AFUB_063940Lysophospholipase 3 B0XZV8 AFUB_034540 Plb3Lysophospholipase 1 B0Y665 AFUB_065820 Plb1Rho GTPase Rho1 B0Y776 AFUB_072830 Rho11,3-Beta-glucanosyltransferase Bgt1 B0XQR5 AFUB_010890 Bgt1Cell wall glycosidase B0XNL0 AFUB_015530 Aspf9/Crf1Cell wall glucanase, putative B0XY72 AFUB_039870 Crh3Mannan polymerase II complex ANP1 subunit Anp1, putative B0XUV6 AFUB_031580 Van1

(Continued on next page)

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detected. Of note, the proteins identified under nonregenerating conditions likelyrepresent underestimations, as a consequence of the lower production of EVs underthose conditions.

DISCUSSION

Yeast forms of different fungal species produce extracellular membrane structuresclassified as EVs (40). More recently, it was demonstrated that filamentous forms offungi also produce EVs (18–20, 22). Although the functional impact of these findings isstill not clear, they confirm that EVs are released by fungi in different morphologicalstages as part of distinct physiological events. In most eukaryotes, EVs are released atthe plasma membrane level. However, in fungi and plants, the cell wall is usually theoutermost cell layer, increasing the complexity of understanding the physiologicalfunction of eukaryotic EVs. Our study has shown that living wall-less stages such asprotoplasts are useful as models to analyze the role of fungal EVs in cell wall biogenesis.Of note, protoplasts of other species, including C. albicans, Schizosaccharomyces pombe,and Neurospora crassa, also produced extracellular particles resembling EVs (23, 27, 41),as concluded from previous microscopic analyses of fungal cells.

Our SEM analysis of protoplast forms of A. fumigatus germinating conidia revealedthe presence of particles with general properties compatible with those of EVs beingreleased from the plasma membrane, including morphology, dimensions, and bilayeredmembranes (42). These vesicular particles were morphologically similar to those ob-served in association with the cell wall of C. neoformans (43). This result and theobservation of vesicles emerging from the A. nidulans surface (23) suggested that A.fumigatus protoplasts are efficient producers of EVs.

EVs released by regenerating protoplasts showed fibril-like material attached to thelipid surface, as revealed by TEM of isolated EVs. Even though the EVs were producedin the highest number in regenerating protoplasts, it is noteworthy that EV release wasnot uniquely associated with cell wall biosynthesis since the nonregenerating proto-plasts also produced EVs. This observation suggests that EV release is not exclusivelyrelated to the synthesis of the cell wall and that the release of EVs in nongrowing cellsmay be also a response of the fungus to extracellular stress such high osmotic pressureor lack of nutrients. It is noteworthy that EVs from cell wall-regenerating cells andnonregenerating protoplasts differed in relative concentrations. Considering that theseexperimental systems correspond to conditions of nutrient abundancy and starvation,respectively, the quantitative differences could simply correspond to a more efficientmetabolic response of nonstarved cells. Indeed, the impact of the nutritional availabilityon the production of microbial EVs has been reported before. In Mycobacteriumtuberculosis, EV production was found to increase in response to iron restriction (44).Alternatively, the increased number of EVs in our protoplast model might indicate anassociation between vesicle production and cell wall synthesis. That supposition re-mains to be experimentally proved, but the finding that S. cerevisiae strains withdeletions in cell wall biosynthesis genes produced more EVs than parental cells (45)argues against this hypothesis but favors the hypothesis that stress increases produc-tion of EVs.

Our carbohydrate analysis revealed the presence of Man and Gal, an increasedamount of Glc, and the presence of GalNAc in EVs obtained from regeneratingprotoplasts. Glc is a marker of �1,3 or �1,3 glucan. Man and GaI are markers of

TABLE 1 (Continued)

Condition UniProt annotationUniProtno. Accession no.

Standardname(s)

SUN domain protein (Uth1), putative B0YCQ5 AFUB_091030 Sun1Probable beta-glucosidase BtgE B0Y9Q9 AFUB_081940 BtgE/Sw11Putative UDP-galactopyranose mutase B0XWU8 AFUB_036480 Ugm1Endo alpha-1,4 polygalactosaminidase, putative B0XYK5 AFUB_041230 Ega3

aFor threshold detection limits and false-discovery rates, please see Materials and Methods.

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galactomannan. Our current results suggest that the EV population produced by A.fumigatus includes plasma membrane-derived vesicles, as consistently described forthe mammalian EVs denominated microvesicles (24). Therefore, we speculate that �1,3or �1,3 glucan is incorporated in the EVs during their formation, as concluded fromprevious demonstrations that cell wall polysaccharides are synthesized on the internalside of the plasma membrane level and extruded in the cell wall at the C-terminalpore-like part of the respective enzymes (46, 47). Galactomannan is assembled in theGolgi apparatus and secreted to the plasma membrane before being cross-linked to�1,3 glucan, supposedly by extracellular transglycosidases (35). Therefore, the possiblepresence of glucans and galactomannan in EVs may be a consequence of their originalassociation with the plasma membrane. GAG, which is a virulence-associated compo-nent of the A. fumigatus extracellular matrix (48, 49), is localized on the surface of thecell wall, where it acts as a component of the fungal extracellular matrix (50). Thedetection of GalNAc residues only in EVs obtained from regenerating protoplastssuggests that GAG is transported by vesicles through the cell wall to be deposited onthe cell surface. Alternatively, GAG could be loosely associated with EVs, considering its“sticky” nature due to its great ability to form unspecific hydrogen bonds. Similarobservations were described in C. neoformans, in which EVs were found to contain theextracellular polysaccharide glucuronoxylomannan (9).

It is still unknown whether the EVs characterized in our protoplast model representthe vesicular structures produced by intact A. fumigatus. In our study, we identified ahigher diversity of EV proteins than of A. fumigatus mycelial vesicles (22), but thehypotheses explaining these differences are numerous. First, it is important to highlightthat the strains used in these studies were distinct, which impairs an accurate com-parison. Second, the proteomic analyses in these independent studies were performed,as usual in the literature, under very different technical conditions. Finally, all studies onfungal EVs produced so far had used distinct conditions for fungal growth that do notcorrelate with our model of nutritional abundancy (cell wall synthesis) or starvation(nonregenerating conditions). Compositional comparisons between this study andothers are, therefore, of very limited applicability. Nevertheless, 32 of the 60 proteinsdescribed for the mycelial A. fumigatus EVs were also found in our study.

Our current results reinforce the idea that biogenesis of fungal EVs includes vesicleformation at the plasma membrane level, as demonstrated for other eukaryotes (24).However, the presence of intracellular sites of vesicle biogenesis and their relationshipwith the synthesis of the cell wall cannot be ruled out. For instance, Neurospora crassachitin synthases 1, 3, and 6 were previously shown to be distributed into cytoplasmicvesicular compartments likely corresponding to chitosomes (51). In Zymoseptoria tritici,chitin and �(1,3)-glucan synthases were previously found to be coexported to the cellsurface within the same vesicle (52). In plant cells, the glucan synthase-like proteinNaGSL1 was detected in both intracellular vesicles and the plasma membrane, thelatter location being associated with cell wall synthesis (53). In the Aspergillus model,protoplasts are produced from the germ tube tips, so they contained all the cell wallsynthesis and remodeling machinery normally present in the plasma membranes offungal apexes (36, 54, 55). Consequently, it is not surprising that protoplast EVscontained many of these cell wall enzymes. Some of these proteins, including Fks1,CsmB and Chs, Gel4, Pmt4, Ktr4, Ktr7, and Gt4C, are essential for the synthesis of themajor cell wall polysaccharides �1,3 and �1,3 glucans, chitin, galactomannan, and GAGand for branching/elongation of the �1,3 glucan in A. fumigatus (56–58). The absenceof GlcNAc (N-acetylglucosamine residues) may suggest that chitin-related moleculesare absent in the EVs. However, we cannot rule out the possibility that a slower kineticsof chitin synthesis affected our experimental model. For instance, preliminary experi-ments of polysaccharide immunolabeling of regenerating protoplasts showed that �1,3glucan was the first polysaccharide detected on the surface of the protoplasts, follow-ing by �1,3 glucan. Chitin was the last polysaccharide to be detected (A. Beauvais,unpublished results).

Proteins that are not predicted to be in the extracellular milieu were abundantly

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detected in EVs from A. fumigatus protoplasts. This observation agrees with numerousreports on the protein composition of fungal EVs (8, 11, 14, 31) and with the fact thatthe biogenesis of these membranous structures has been linked to the cytoplasm andthe plasma membrane (59). Additionally, it was previously suggested that the cell wallis a storage site for many fungal proteins, including glucanases, PhiA, Ecm33, Gel1, andGel4 (60–63).

Our current results contribute to a better understanding of the properties of fungalEVs. To our knowledge, this is the first characterization of EVs in protoplasts obtainedfrom germinating conidia. Our results suggest that these cellular forms represent apromising model to explore novel roles of fungal EVs in many fungal species. In the A.fumigatus model, phagocytic cells stimulated with EVs increased their ability to produceinflammatory mediators and to promote fungal clearance (22). Similarly, A. flavus EVsaffected the interaction between the fungus and host immune cells (64). These resultssupport the relevance of the use of protoplastic fungal EVs to promote better under-standing of their role in both the physiology and immunopathogenesis of A. fumigatus.

MATERIALS AND METHODSGrowth conditions and preparation of A. fumigatus protoplasts. The A. fumigatus reference strain

used in this study was CEA17ΔakuBKU80 (ku80), which is deficient in nonhomologous end joining (65). TheCEA17ΔakuBKU80 strain was conserved on 2% (wt/vol) malt agar slants. Five-day-old conidia wererecovered from the slants by vortex mixing performed with a 0.05% (vol/vol) aqueous Tween 20 solutionand filtered through a 40-�m-pore-size cell strainer. For protoplast preparation, 7 � 109 conidia sus-pended in 0.05% Tween were centrifuged at 3,000 � g for 10 min. The supernatant was discarded, andthe cells were suspended in 10 ml of sterile water. The conidia were inoculated in 600 ml of germinationmedium (1% yeast extract, 3% glucose, and 0.6 M mannitol) and then incubated with shaking for 14 hat 30°C. Germinated conidia were harvested and separated from dormant conidia by filtering the samplethrough a sterile Miracloth-lined funnel and then washed with 200 ml of sterile osmotic medium (OM;1.2 M MgSO4 · 7H2O, 0.09 M K2HPO4, and 0.01 M KH2PO4, pH 5.8) and subjected to 2-fold dilution. Afterthe washing step, the germinated conidia were suspended in 20 ml OM containing Glucanex (NovoNordisk Ferment Ltd., catalog number CH4243) at 30 mg/ml. The cells were gently homogenized, and thesuspension was adjusted to a final volume of 250 ml with a filtered-sterile Glucanex solution forhydrolysis of the cell wall.

Cell wall digestion was performed in 2-liter Erlenmeyer flasks for 2 h at 37°C, with gentle shaking(60 rpm), until sufficient protoplasts were released, as assessed microscopically. For protoplast recovery,the cells were harvested by filtration in a sterile glass Buchner funnel (porosity 2). After filtration, 2volumes of sterile 0.3 M KCl were added to 1 volume of the protoplast suspension in Glucanex. Themixture was centrifuged at 5,000 � g (20 min, 25°C). The pellet was suspended in sterile 0.6 M KCl andwashed twice (3,000 � g, 10 min per wash, 25°C, with minimal break) to eliminate the remainingGlucanex. The final pellet of fresh protoplasts was divided into two equal parts. Each part was suspendedin 10 ml of sterile 0.6 M KCl and further incubated under nonregenerating or cell wall synthesisconditions. The viability of the protoplasts was assessed by the use of trypan blue dye exclusion atdifferent time points, from 0 to 2 h.

For cell wall regeneration, the protoplasts were incubated in 400 ml of a minimal medium (MM)supplemented with 0.6 M KCl for 2 h at 37°C, with shaking (120 rpm). The MM was prepared as previouslydescribed (66, 67) with some modifications and contained 1% (wt/vol) glucose, 20 mM glutamine, 0.052%KCl, 0.052% MgSO4 · 7H2O, 0.152% KH2PO4, and 1 ml trace element solution (pH 6.5). Alternatively,protoplasts were incubated for 2 h in a nonregenerating solution of 0.6 M KCl or were immediatelyprocessed for the analyses described below.

Microscopic analysis of protoplasts. Freshly purified protoplasts, as well as protoplasts obtainedunder cell wall-regenerating or nonregenerating conditions, were fixed with 2% formaldehyde– 0.6 M KCland stored at 4°C. For fluorescence or superresolution microscopy, the cells were washed twice withphosphate-buffered saline (PBS) and blocked with superblock blocking buffer (Thermo Scientific, catalognumber 37515) mixed in PBS for 1 h at 37°C. Surface �-(1,3)-glucan of protoplasts was labeled with MOPC104E monoclonal antibody (Sigma-Aldrich, catalog number M5909) (2 �g/ml; 1 h at 37°C) (68). Alterna-tively, the cells were stained with an anti-GAG monoclonal antibody (20 �g/ml; 1 h at 37°C) produced inthe Latgé laboratory as previously reported (48). After two washes in PBS, each preparation wasincubated with the appropriate secondary antibodies (anti-mouse IgM Alexa Fluor 488 for glucanstaining; anti-mouse IgG Alexa Fluor 488 for GAG; both diluted 1:100 in blocking buffer). After incubationfor 1 h at 37°C, the cells were washed three times with PBS. The protoplast membranes were finallystained with Vybrant DiI cell-labeling solution (Molecular Probes, catalog number V22885) at 5 �M(30 min, 37°C) and washed one final time with PBS. The cells were placed on glass slides covered withProLong Gold antifade reagent. The cells were microscopically observed under a Zeiss Axioplan 2fluorescence microscope or a Zeiss Elyra PS.1 superresolution microscope using structural illuminationmode. Images were obtained with ZEN 2.1 software.

Superresolution scanning electron microscopy (SEM) was performed as previously described (69).Briefly, 106 protoplasts were fixed on sterile glass coverslips (previously coated with poly-L-lysine)

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overnight at 4°C on 24-well plates. The samples were dehydrated in a graded ethanol series (30%, 50%,and 70% for 5 min and 95% and 100% for 10 min), subjected to critical point drying in CO2, mounted onstubs, and coated with carbon. Observation of the protoplast cell surface was performed with an Auriga40 field emission scanning electron microscope (FE-SEM) microscope (Zeiss, Germany).

Protoplasts were also were analyzed with a JEOL JSM-6700F apparatus, which is an ultra-high-resolution field emission scanning electron microscope equipped with a cold-field-emission gun and astrongly excited conical lens. The secondary-electron image resolution settings were 1 nm at 15 kV and2.2 nm at 1 kV. Pieces of culture were frozen using a Gatan Alto 2500 cryo-stage and cryo-preparationchamber. The preparation conditions were as described previously by Paris et al. (70).

EV isolation and physical-chemical analysis. Isolation of EVs from protoplast supernatants wasperformed as previously described for yeast cells (40), with minor modifications. Briefly, after eachincubation period, the supernatants were separated from the protoplast cells by centrifugation at3,000 � g (15 min, 25°C, with no brake) and sequentially passed through filters with 5-�m, 1.2-�m, and0.45-�m pore sizes. The pellets containing protoplast cells were stored at �20°C for sterol quantification.After filtration, the supernatants were concentrated in an Amicon ultrafiltration system (cutoff, 100 kDa)and again centrifuged at 10,000 � g and 4°C for 15 min to eliminate possible cellular debris. Theconcentrated supernatants were finally ultracentrifuged at 100,000 � g (4°C, 1 h). The resulting pelletscontaining EVs were washed twice with filtered PBS (0.22-�m pore size) under the same ultracentrifu-gation conditions and finally suspended in 300 �l of filtered PBS (0.22-�m pore size). The EV suspensionswere stored at �80°C for further experiments.

For nanoparticle tracking analysis (NTA) and GAG serological detection, the EV suspensions were firstsubmitted to immunoprecipitation for the removal of nonvesicular polysaccharides. In this assay, 50 �lof the EV suspension was added to the wells of a 96-well enzyme-linked immunosorbent assay (ELISA)plate, previously coated with a mixture of antibodies against �-glucan (J558) (71), �-glucan [Dectin 1human IgG Fc chimeric ��(1,3)�glucan receptor; a kind gift of G. Brown, University of Aberdeen, UnitedKingdom], and GAG (1 �g/ml, 1 h, 37°C) (48) and blocked with PBS containing 1% bovine serum albumin(BSA). Unbound fractions were collected, and the resulting EVs were stored at �80°C for furtherexperiments.

Nanoparticle tracking analysis (NTA) was performed to determine the EV diameter and concentration.NTA of protoplast EVs was performed on an LM10 nanoparticle analysis system coupled with a488-nm-wavelength laser and equipped with a scientific complementary metal oxide semiconductor(sCMOS) camera and a syringe pump (Malvern Panalytical, Malvern, United Kingdom), as recentlydescribed for C. gattii EVs (72). The samples were subjected to 25-fold dilution in filtered PBS andmeasured within the optimal dilution range of 7.6 � 107 to 6.8 � 108 particles/ml. The data wereacquired and analyzed using NTA 3.0 software (Malvern Panalytical). NTA values were used to calculateratios of EVs to cells by dividing the number of particles detected in the equipment by the number ofcells in the original individual vesicle samples. For these analyses, two independent biological replicateswere prepared and each sample was submitted to at least three reads, generating six measurements foreach sample. These values were adjusted according to the original sample dilution (25-fold). Thequantification of sterol in EV preparations was performed with an Amplex red cholesterol assay kit (14,73, 74).

Transmission electron microscopy (TEM) of EVs. For negative-staining TEM, the EV pellets werefixed with 2% glutaraldehyde–2% paraformaldehyde– 0.1 M sodium cacodylate buffer at room temper-ature for 2 h and then postfixed overnight at 4°C with 1% glutaraldehyde– 4% paraformaldehyde–PBS.Copper carbon-coated grids (Cu-CF300; EMS), previously negatively charged by the use of an Elmosystem (1 min, 15 pascals, 2 mA, 80 V), were put in contact with 15 �l of each sample for 10 min andwashed three times with Milli-Q water drops (2 min each time), stained with uranyl acetate 2%, dried, andobserved with a Tecnai Spirit microscope operating at 120 kV and equipped with an Eagle 4,000-pixel-by-4,000-pixel camera.

EVs were alternatively fixed with 2% formaldehyde–2% glutaraldehyde– cacodylate buffer (0.1 M, pH7.4). The samples were washed through four changes of cacodylate buffer (30 min each) and pelleted in1% agarose (JT Baker Chemical Co., Phillipsburg, NJ). They were transferred to 1% osmium tetroxide incacodylate buffer (0.1 M, pH 7.4) and incubated at 4°C for 1 h followed by washing in cacodylate bufferand distilled water performed for a total of 30 min. The samples were then stained with 0.5% aqueousuranyl acetate, dehydrated, slowly infiltrated with epoxy, and embedded. After resin polymerization, theblocks were sectioned on a Leica ultramicrotome and subjected to poststaining for 10 min in 2% uranylacetate–50% ethanol and for 5 min in lead citrate. Ultrathin sections (70 nm thick) were collected onFormvar-coated copper slot grids and subjected to poststaining for 10 min in 2% uranyl acetate–50%ethanol and for 5 min in lead citrate. Sections were then examined on a JEOL 1200EX microscope (JEOLLtd., Tokyo, Japan) equipped with a SIA L3C charge-coupled-device (CCD) camera (SIA Inc., Duluth, GA).

Monosaccharide composition and serological detection of GAG in EVs. EV ultracentrifugationpellets were suspended in water for monosaccharide analysis. The presence of monosaccharides in theEVs was determined by gas chromatography after hydrolysis, reduction, and paracetylation of the vesiclecomponents using meso-inositol as an internal standard (75). Serological estimation of vesicular GAGlevels was performed as described before by our group for other fungal polysaccharides (74). Briefly, EVsuspensions were vacuum dried and suspended in chloroform/methanol (9:1 [vol/vol]). The suspensionwas centrifuged, and the resulting white precipitate was solubilized in PBS for quantitative ELISAperformed with the anti-GAG antibody. The purified polysaccharide (SGG; a kind gift of T. Fontaine,Institut Pasteur, Paris, France) was used for the preparation of standard curves and determination ofpolysaccharide concentrations in EV samples (48).

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Protein composition of EVs. EV ultracentrifugation pellets were suspended in buffer containing 8 Murea–100 mM Tris (pH 7.5) for proteomic analysis. Protein samples were reduced with 5 mM dithiothreitol(DTT) for 30 min at 23°C and then alkylated with 20 mM iodoacetamide in the dark at room temperature,for 30 min. Subsequently, the endoproteinase LysC (Promega) was added for the first digestion step(protein-to-Lys-C ratio � 80:1) for 3 h at 30°C. The sample was then diluted to reach a 1 M ureaconcentration with 100 mM Tris (pH 7.5), and trypsin (Promega) was added to the sample (protein-to-trypsin ratio � 50:1). The samples were digested for 16 h at 37°C. Proteolysis was stopped by the additionof 1% formic acid (FA). The resulting peptides were desalted using a Sep-Pak SPE cartridge (Waters)according to the manufacturer’s instructions.

Analysis of digested peptides by the use of liquid chromatography coupled to tandem massspectrometry (LC-MS/MS) was performed on an Orbitrap Q Exactive Plus mass spectrometer (ThermoFisher Scientific, Bremen, Germany) coupled to an EASY-nLC 1000 liquid chromatograph (Thermo FisherScientific). The peptides were loaded and separated at 250 nl · min�1 on a homemade C18 50-cm capillarycolumn with a picotip silica emitter (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) (75-�mdiameter filled with 1.9-�m-pore-size Reprosil-Pur Basic C18-HD resin) equilibrated in solvent A (0.1%formic acid). The peptides were eluted using a gradient of solvent B (acetonitrile [ACN], 0.1% FA) of from2% to 18% for 110 min, 18% to 30% for 35 min, and 30% to 45% for 15 min under conditions of a 250nl/min flow rate. The total duration of the chromatographic run was 185 min, including high-ACN-levelsteps and column regeneration. Mass spectra were obtained in data-dependent acquisition mode withXcalibur 2.2 software (Thermo Fisher Scientific, Bremen) with automatic switching between MS andMS/MS scans using a top-10 method. Spectral resolution corresponded to 70,000 (at m/z 400) with atarget value of 3 � 106 ions. The scan range was limited from 300 to 1,700 m/z. Peptide fragmentationwas performed via higher-energy collision dissociation (HCD), with the energy set at a normalizedcollision energy (NCE) value of 28. The intensity threshold for the ion selection was set at 1 � 106 ions,with charge exclusion settings of z � 1 and �7. The MS/MS spectra were acquired at a resolution of17,500 (at m/z 400). The isolation window was set at 1.6 Th. Dynamic exclusion was employed within 45 s.

A data search was performed with MaxQuant tool (76) (version 1.5.3.8) with the Andromeda searchengine against the A. fumigatus A1163 database (9,942 entries, downloaded from https://www.uniprot.org [accessed 18 September 2019]). As search parameters, carbamidomethylation of cysteines was setas a fixed modification and oxidation of methionine and protein N-terminal acetylation were set asvariable modifications. The mass tolerances in MS and MS/MS were set to 5 ppm and 20 ppm, respec-tively. The maximum peptide charge value was set to 7, and 7 amino acids were required as theminimum peptide length. A false-discovery rate of 1% was set for both protein and peptide levels. Fourindependent EV ultracentrifugation pellets were analyzed for protoplasts submitted either to cellwall-regenerating conditions or repressing conditions. Two EV samples of freshly purified protoplastswere analyzed. Proteins identified in at least two independent experiments were assigned for GeneOntology (GO). GO term enrichment was performed using the GOtermfinder feature of the AspGDdatabase (http://www.aspergillusgenome.org/cgi-bin/GO/goTermFinder). Enriched GO terms were sum-marized by removing redundancy using the REVIGO Web server, available at http://revigo.irb.hr/ (77).Protein identification was based on the detection of one representative peptide by mass spectrometryafter protein digestion. For sample preparation and qualitative comparison, EV samples were obtainedfrom cultures with similar inocula and volumes. Revigo outputs were viewed in R (v4.0.0) using thetreemap package. Treemap construction was based exclusively on proteins shared by all replicates.

Statistical analysis. All statistical analyses were performed using GraphPad Prism 6 software(GraphPad Software Inc.). Data sets were tested for normal distribution using Shapiro-Wilk orKolmogorov-Smirnov normality tests. In the cases in which the data passed the normality test (al-pha � 0.05), they were further analyzed using the unpaired Student’s t test. Multiple data sets werefurther analyzed using ordinary one-way analysis of variance (ANOVA), followed by the Tukey’s multiple-comparison test. When at least one data set was nonnormally distributed, multiple data sets wereanalyzed by the nonparametric Kruskal-Wallis test.

Data availability. Accession numbers for proteins described in this work are available in Table 1. Weconfirm that the data supporting the findings of this study are available within the article and itssupplemental material. Additional data supporting our findings are available from J.R. or M.L.R. uponreasonable request.

SUPPLEMENTAL MATERIALSupplemental material is available online only.FIG S1, PDF file, 2.4 MB.TABLE S1, XLSX file, 0.7 MB.

ACKNOWLEDGMENTSM.L.R. was supported by grants from the Brazilian Ministry of Health (grant 440015/

2018-9), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grants405520/2018-2 and 301304/2017-3), and Fiocruz (grants VPPCB-007-FIO-18 and VPPIS-001-FIO18). We also acknowledge support from the Instituto Nacional de Ciência eTecnologia de Inovação em Doenças de Populações Negligenciadas (INCT-IDPN). J.R.developed part of this work as a Ph.D. student supported by FAPERJ (E-26/201.991/

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2015) and is currently a postdoctoral fellow at Institut Pasteur (Paris, France), funded bythe CAPES-Cofecub Program (88887.142840/2017-00). This research was partly sup-ported by funds provided to J.-P.L. by the Laboratoire d’Excellence “Integrative Biologyof Emerging Infectious Diseases” (grant ANR-10-LABX-62-IBEID), la Fondation pour laRecherche Médicale (DEQ20150331722 LATGE Equipe FRM 2015).

We are grateful to Leandro Honorato (Instituto de Microbiologia Paulo de Goes,UFRJ, Brazil) and Luna Sobrino Joffe (Stony Brook University, USA) for help withprotoplast purification, Flavia Reis (Fiocruz, Brazil) for help with NTA, Fernando P. deAlmeida for the help with superresolution microscopy (CENABIO/UFRJ, Brazil), andVishukumar Aimanianda (Institut Pasteur, France) for discussions and for providingsome of the antibodies for this study.

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